Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.
Fermentation is a process in which yeast metabolise simple sugars in fruit or vegetable matter primarily into ethyl alcohol and carbon dioxide. This process is involved in the production of a variety of comestible products such as bread, beer, wine, liquor, surface-ripened cheese and vinegar as well as in the production of biofuels, vitamins, and enzymes.
Wine is a product which is in part produced by alcoholic fermentation of grape juice by yeast. The yeast strains present in the fermentation process metabolise grape juice carbohydrates into ethanol and carbon dioxide. However, these components make a relatively minor contribution to the overall wine flavour. The aroma and flavour characteristics of wine result from a large number of compounds, including volatile acids and carbonyl compounds, 400 of which have been identified as being produced by yeast during the fermentation process (Nykänen, 1986).
Fermentation may proceed spontaneously, where the suite of microbes naturally present conduct the ferment; alternatively a winemaker may deliberately inoculate the grape juice with a large culture of a known commercially available wine yeast. The principle yeast used to inoculate grape juices are various strains of Saccharomyces cerevisiae. 
The initial stages of spontaneous wine fermentation generally involve the growth of non-Saccharomyces yeasts, which exhibit low fermentative properties and thrive under a low alcohol milieu. These yeasts include Hanseniaspora (Kloeckera) and Candida (eg Candida stellata, C. pulcherrima) (Heard and Fleet, 1986) as well as other species. In the spontaneous case, fermentation involves the sequential interaction of a variety of yeast genera and species (Heard and Fleet, 1988).
The sensitivity of non-Saccharomyces yeasts to environments of over 5% ethanol means that their growth is usually limited to the first 2 to 3 days of wine fermentation, following which the ethanol-tolerant Saccharomyces yeast become dominant. Importantly, the non-Saccharomyces strains can influence the fermentation significantly as they can reach populations of 106-107 cells/ml (Lema et al., 1996). This influence includes the production of a wide range of volatile and non-volatile products such as organic acids, higher alcohols and esters that contribute substantially to the aroma and flavour characteristics of the wine (Rapp and Versini, 1991; Esteve-Zarzoso et al., 1998). The nature and concentrations of these “flavour compounds” are determined by the genera and species of the yeast present in the fermentation (Houtman et al., 1980; Lambrechts and Pretorius, 2000; Brandolini et al., 2002; Ramano et al., 2003). There is an increasingly large body of work examining the contribution of non-Saccharomyces species to wine flavour and aroma (Esteve-Zarzoso et al., 1998). Some of this work shows that mixed species ferments produce unexpectedly high levels of some flavour-active compounds including, for example, esters (Garde-Cerdan and Ancin-Azpilicueta, 2006; Rojas et al., 2003) and 2,3-butanediol (Clemente-Jiminez et al., 2004).
Volatile thiols are a family of flavour compounds found in wine (Gachons et al., 2000). They include, for example, 4-mercapto-4-methyl-pentan-2-one (4MMP), 4-mercapto-4-methyl pentan-2-ol (4MMPOH), 3-mercaptohexan-1-ol (3 MHA) and 3-mercaptohexyl acetate (3 MHA). Some of these thiols, eg. 3 MH and 3 MHA, have distinctive grapefruit- and passionfruit-like aromas and flavours (Tominaga et al., 1998a).
3 MH and 3 MHA are often found in Sauvignon Blanc (SB) wines at concentrations above the human sensory threshold. A cysteine-conjugated, non-volatile, flavourless precursor of 3 MH, S-(3-hexan-1-ol)-cysteine, is found in SB grape juice, and during fermentation this precursor is potentially cleaved to liberate 3 MH, which is subsequently converted to 3 MHA (Gachons et al., 2000). The enzymes necessary for the processing of these precursors are thought to derive from yeasts, and conversion requires active yeast growth (Tominaga et al., 1998b). The enzymes involved are possibly some form of β-lyase and acetyltransferase respectively. One publication has suggested that there are multiple yeast genes encoding β-lyase enzymes (Howell et al., 2005). However, scientists at two laboratories, including the present inventors, have been unable to repeat these results. To date, we consider that no robust data showing the precursors, enzymes or pathways responsible for the liberation of thiols by yeast have been published.
Chr Hansen (Denmark) produce four commercially available yeast starter cultures that include mixtures of different yeast species called Harmony, Symphony, Rhythm, and Melody. The yeast species included in these mixes are S. cerevisiae, Torulaspora delbrueckii and Kluyveromyces thermotolerans. The mixes have been reported to produce wines with an altered flavour profile but there has been no suggestion that the flavour profile is related to the presence, mix or concentration of thiols.
No studies have examined the effects of non-Saccharomyces species on the level of volatile thiols in wine or of the effects of mixtures of yeast species on thiol production or wine flavour. We are aware of only one study in which the effect of a Saccharomyces species on the level of volatile thiols in wine was measured. This related to the measurement of 4MMP produced by Saccharomyces cerevisiae in wine (Howell et al. 2004).
An understanding of the biological processes that control the presence and amount of compounds in a fermentation process is highly desirable. In particular, being able to regulate the amount and type of thiols in wine would be very beneficial to winemakers.
Regulation of the amount of thiols, and in particular 3 MH and 3 MHA, in wine would allow for the development of new technologies permitting winemakers to more precisely alter the amounts of these distinctive flavour compounds in their product. Such a technology would, therefore, be of significant commercial value.
It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.